Several reports have appeared recently in the literature detailing cases in which gastrointestinal disease, often with significant associated morbidity, occurred in acquired immune deficiency syndrome patients infected with microsporidial parasites. As a consequence of these reports, there has been an increased awareness among clinicians of the potential importance of microsporidia as gastrointestinal (GI) pathogens. Attention is now being focused on methods for both diagnosis and treatment. There is considerable interest in determining the diagnostic value of stool specimens for detecting GI disease due to microsporidia. A variety of staining modalities have been evaluated for their ability to visualize microsporidia in stool preparations. Unfortunately, the various species of microsporidia that infect humans can be identified accurately only by visualizing characteristic subcellular morphologic features of organisms in electron micrographs of tissue specimens. Because the efficacy of treatment regimens currently in development may be somewhat species specific, and because invasive procedures are necessary to obtain appropriate specimens for electron microscopy (EM), a more widely available approach to identification than EM is clearly required. We have published a report that describes our polymerase chain reaction (PCR) assay for amplification of microsporidial rRNA genes followed by restriction endonuclease digestion of PCR products as a method for identifying microsporidia in fresh stool. The PCR primers used in the assay will amplify all of the currently identified microsporidial human pathogens. We are continuing to modify the assay to allow detection of microsporidia in formalin-fixed stool specimens. A Southern blot format using PCR-amplified DNA and species-specific DNA probes has been developed for use in microsporidial speciation. The final stage of the project will be to determine the sensitivity of our assay and the length of time specimens can be stored in formalin and still allow detection of the microsporidia. We have developed a shorter, cheaper, and less rigorous method for extraction of microsporidial DNA from stool.